title: Cloning and characterization of 2S albumin, Car i 1, a major allergen in pecan.
abstract: Although pecans are associated with IgE-mediated food allergies, the allergens responsible remain to be identified and characterized. The 2S albumin gene was amplified from the pecan cDNA library. Dot-blots were used to screen the recombinant protein with pecan allergic patients' serum. The affinity purified native protein was analyzed by Edman sequencing and mass spectrometry/mass spectrometry (MS/MS) analysis. Cross-reactivity with walnut was determined by inhibition enzyme-linked immunosorbent assay (ELISA). Sequential epitopes were determined by probing the overlapping peptides with three different patients' serum pool. The 3-dimensional homology model was generated, and the locations of the pecan epitopes were compared with those of known sequential epitopes on other allergenic tree nut homologues. Of 28 patients tested by dot-blot, 22 (79%) bound to 2S albumin, designated as Car i 1. Edman sequencing and the MS/MS sequencing of native 2S albumin confirmed the identity of recombinant (r) Car i 1. Both pecan and walnut protein extracts inhibited the IgE-binding to rCar i 1. Sequential epitope mapping indicated weak, moderate, and strong reactivity against 12, 7, and 5 peptides, respectively. Of the 11 peptides recognized by all serum pools, 5 peptides were strongly reactive and located in 3 discrete regions of the Car i 1 (amino acids 43-57, 67-78, and 106-120). Three-dimensional modeling revealed IgE-reactive epitopes to be solvent accessible and share significant homology with other tree nuts providing a possible basis for previously observed cross-reactivity.
authors.affiliation: Department of Nutrition, Food and Exercise Sciences, The Florida State University, Tallahassee, Florida 32306, United States.
authors.forename: Girdhari M
authors.initials: GM
authors.lastname: Sharma
authors.forename: Andre
authors.initials: A
authors.lastname: Irsigler
authors.forename: Pushparani
authors.initials: P
authors.lastname: Dhanarajan
authors.forename: Rosalia
authors.initials: R
authors.lastname: Ayuso
authors.forename: Luda
authors.initials: L
authors.lastname: Bardina
authors.forename: Hugh A
authors.initials: HA
authors.lastname: Sampson
authors.forename: Kenneth H
authors.initials: KH
authors.lastname: Roux
authors.forename: Shridhar K
authors.initials: SK
authors.lastname: Sathe
mesh_terms: D000418:Albumins / Q000235:genetics*; D000485:Allergens / Q000235:genetics*; D001483:Base Sequence; D031325:Carya / Q000235:genetics / Q000276:immunology*; D002846:Chromatography, Affinity; D003001:Cloning, Molecular; D003429:Cross Reactions; D017931:DNA Primers; D018744:DNA, Plant; D004797:Enzyme-Linked Immunosorbent Assay; D018604:Epitope Mapping; D008969:Molecular Sequence Data; D053719:Tandem Mass Spectrometry
chemical_list: D000418:Albumins; D000485:Allergens; D017931:DNA Primers; D018744:DNA, Plant